One of the protagonists, Rey, keeps precious cargo with her as she searches for Luke: the lightsaber of Anakin Skywalker, Luke’s father. She must deliver the lightsaber to Luke in order to galvanize his joining the Resistance and to help motivate him to resume his role as a Jedi Master.

Just like how the galaxy would be doomed if Rey could not reach Luke, budding yeast cells would be doomed if a Hsp70 protein chaperone could not interact with another chaperone, Hsp90. But Hsp70 isn’t trying to deliver a lightsaber to Hsp90—instead, Hsp70 is trying to deliver “client” protein substrates, so that Hsp90 can help fold and mature these proteins.

In Star Wars, the trusty droid R2-D2 was ultimately there to help Rey find Luke with a holographic map to Luke’s location. But is our friend Saccharomyces cerevisiae just as fortunate? In fact, it just so happens that S. cerevisiae also has an “R2-D2” of its own: the co-chaperone Sti1p. Just like how R2-D2 helps Rey find Luke, Sti1p helps bring Hsp70 and Hsp90 together so that Hsp90 can receive its protein substrates and save the galaxy (or at least help fold proteins and save the yeast cell).

To give some background, Hsp90 (encoded by HSP82 and HSC82 in yeast) is a molecular chaperone that assists in the folding and maturation of specific protein substrates, or “clients”. It functions as a homodimer that undergoes ATP-regulated cycles of “opening” up to receive clients, closing, and then opening again. Many clients of Hsp90 first bind to a chaperone of the Hsp70 family, such as Ssa2p, which assists in the early stages of protein folding before interacting with Hsp90 and passing on the client.

The co-chaperone Sti1p comes into the picture by bridging Hsp70 and Hsp90 and helping them interact, so that the Hsp70-bound client can be delivered to Hsp90. Although this function of Sti1p has long been known, the exact mechanistic details have been obscure, and mutational studies have suggested that Sti1p does more than just bridge the two chaperones together.

Thanks to a recent GENETICS study by Reidy and coworkers, we now know more about how Sti1p helps save the galaxy: it not only helps Hsp90 interact with Hsp70, but also prepares Hsp90 to receive its client protein and advance in its reaction cycle.

To uncover the role of Sti1p in the Hsp90 cycle, the authors examined Hsp90 mutants that were dependent on Sti1p for viability. They mapped both previously-known and newly found Sti1-dependent mutants and found that all of the mutations clustered in just two sites. They designated the sites Sti1-dependent N and C-terminal domain proximal, or “SdN” and “SdC”, respectively.

Because previous studies showed that some Hsp90 SdN mutants don’t interact well with Hsp70, and that analogous SdC mutations in E. coli weaken interactions with Hsp90 client proteins, the authors hypothesized that Sti1p assists Hsp90 with these functions in particular.

To clarify how Sti1p and Hsp90 cooperate, the authors utilized a combination of mutational studies, pull downs with purified proteins, mass spectrometry, and more. They observed that SdN mutations in Hsp90 reduce interactions with Hsp70, while SdC mutations do not. Further, they found that the Sti1p dependency of SdN mutants could be cured through a novel suppressor mutation (E402R), which increases the interaction of Hsp90 with Hsp70. These results suggest that Hsp90 interacts with Hsp70 through the SdN region, and that Sti1p is needed to bring the two chaperones together if they aren’t able to do so well enough on their own.

Importantly, although the E402R suppressor mutation was able to “cure” SdN mutants of their Sti1p dependency, it was unable to do so in SdC mutants. This indicated that SdC mutants are defective in a function that Sti1p assists with, but one that’s separate from having Hsp70 interact with Hsp90.

To uncover why SdC mutants depend upon Sti1p for viability, the authors investigated suppressor mutations. The authors isolated multiple SdC suppressors and also found that a previously characterized mutation, A107N, is able to ameliorate the effects of SdC mutations. Previous studies on A107N show that this mutation promotes closure of the open-state Hsp90 heterodimer, which is an important step of the Hsp90 reaction cycle. The authors found that other SdC suppressor mutations were consistent with A107N and could relieve the Sti1p dependence of SdC but not SdN mutants. These results indicate that Sti1p not only promotes Hsp90-Hsp70 interaction, but also has an additional function in promoting Hsp90 heterodimer closure and progression of the Hsp90 cycle.

So it turns out that Sti1p is like R2-D2 in more ways than one. In its first role, Sti1p helps Hsp70 and Hsp90 interact, much like how R2-D2 helps Rey find Luke in The Force Awakens. In its second role, Sti1p helps Hsp90 accept its substrates, progress through its reaction cycle, and perform its function. This is similar to what R2-D2 does in Star Wars: The Last Jedi. In the movie, Luke initially shows reluctance to resume his role as a Jedi Master and help save the galaxy, despite being found by Rey. But thankfully, R2-D2 was there to motivate Luke to return to his heroic duties, much like how Sti1p is there to “motivate” Hsp90 to capture client proteins and do its job.

Thanks to the efforts of Reidy and coworkers, how Sti1p helps save the galaxy yeast cell is that much clearer. Not only does Sti1p help Hsp70 interact with Hsp90 and deliver lightsabers client proteins, but it also helps Hsp90 do its job as a Jedi Master chaperone by promoting progression of the Hsp90 reaction cycle!

Unlike the proteins in this egg, most aggregated yeast proteins get back to their normal shape after a heat shock. Image from Wikimedia Commons.

Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.

Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.

When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.

A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.

Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.

In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.

The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.

Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)

After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?

To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.

Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.

After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.

It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.

Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.

None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.

Some ribosomal proteins need to be closely chaperoned even as they’re being born. Image courtesy of the National Library of Medicine via Wikimedia Commons

Some kids are born troublemakers, wreaking havoc and destruction everywhere they go. They can’t help themselves; it’s in their nature to be that way. But if they have concerned and protective adults in their lives, children can overcome this tendency and grow up to become productive members of society.

Within the cell, ribosomal proteins are problem children. Although they grow up to have essential and productive roles, as newborns they can cause big trouble.

Many of them have highly charged, unstructured regions that give them a tendency to aggregate with other proteins. And they have a complicated journey to adulthood, since ribosome assembly happens in multiple cellular compartments. With an estimated 160,000 ribosomal proteins synthesized every minute in rapidly growing S. cerevisiae cells, these troublemakers could cause major problems if left to their own devices.

To help control this unruly mob, certain proteins in the cell act as designated chaperones for ribosomal proteins. In a new paper in Nature Communications, Pausch and colleagues found that, surprisingly, these specialized nurses catch their client proteins even as they’re being born. They swaddle them from the first moment they start to emerge as nascent proteins, and keep them from causing any harm until they can be delivered safely to their final destination.

The researchers first looked at the proteins Rrb1 and Sqt1. Previous work had suggested they might act as specific chaperones for the ribosomal proteins Rpl3 and Rpl10, respectively. And Pausch and colleagues confirmed these results, showing that TAP-tagged Rrb1 pulled down only Rpl3 and Sqt1 only pulled down Rpl10. Each of these troublesome tots had its own personal chaperone!

But surprisingly, very little of the protein was needed for these chaperones to keep ahold of their respective charges. When the authors trimmed down the ribosomal proteins to shorter and shorter lengths, they saw that just the N-terminal 15-20 amino acids of each ribosomal protein were necessary and sufficient for interaction with its chaperone.

They decided to use X-ray crystallography to look in detail at the Sqt1-Rpl10 interaction. First they determined the crystal structure of Sqt1 on its own, and found that it forms an eight-bladed WD-repeat beta-propeller, looking much like a round electric fan. The amino acids positioned on the surface of the blades are negatively charged.

Next, the authors co-crystallized Sqt1 with a peptide corresponding to amino acids 2-15 of Rpl10. The structure showed that the positively charged peptide was cradled in the negatively charged surface.

To test whether these charged residues were important for the interaction, they mutated the charged residues of Sqt1 and of the peptide and combined them in various ways. Sure enough, changing the charged residues of either partner disrupted or diminished the interaction.

Pausch and colleagues went on to test whether those same charged residues are important in vivo. An sqt1 mutation changing glutamate residue 315 to lysine (E315K), that abolished the Sqt1-Rpl10 interaction in vitro, was lethal for yeast cells, confirming the importance of the interaction.

The researchers also detected many allele-specific genetic interactions between the charged residues of the two proteins, and even found that switching the charges in an interacting pair of amino acids (changing an Sqt1 residue to a positive charge and its Rpl10 binding partner to a negative charge) would improve growth compared to either single mutant.

The lethality of that sqt1-E315K mutation, and even the lethality of an sqt1 null mutation, were weakly suppressed by overproduction of Rpl10. So yeast cells can get by (just barely) with an un-chaperoned Rpl10, as long as there’s enough of it around. This result also confirmed that Rpl10 is the only client of Sqt1.

As yet another verification that Sqt1 acts as a chaperone, the authors looked to see what happens to the Rpl10 protein in sqt1 mutants. If cells carrying wild-type SQT1 are lysed and separated into a pellet and supernatant, most Rpl10 spins down in the pellet but a significant amount is soluble in the supernatant. However, if the cells carry any of several sqt1 mutant alleles that alter the charged residues and diminish the interaction with Rpl10, all of the Rpl10 is found glommed together in the pellet.

The two chaperone-ribosomal protein interactions that Pausch and colleagues investigated, Sqt1-Rpl10 and Rrb1-Rpl3, both involved the extreme N termini of the ribosomal proteins. Previous studies had also shown that two other chaperones for ribosomal proteins, Yar1 and Syo1, also interact with the N termini of their clients. So the authors wondered whether interactions between ribosomal proteins and their chaperones might even start during translation of the ribosomal proteins.

In a final experiment, the researchers treated yeast with cycloheximide to freeze translation and then pulled down each of the four chaperones via affinity tags. Each chaperone specifically pulled down the mRNA encoding its client protein, showing that it was binding to the nascent protein as it first started to emerge from the translating ribosome.

So this study has defined a new step in ribosomal biogenesis. Certain specific ribosomal proteins are such troublemakers that it’s too dangerous for the cell to just release them into the cytoplasm after they’re translated.

Instead, these bouncing baby proteins are caught by their individual nurses before they’re even fully born, and wrapped up to protect both the ribosomal proteins themselves and the rest of the cell. Since ribosomal biogenesis is highly conserved across species and since defects in it are associated with many different diseases, further study of these cellular midwives could have important implications for human health. Perhaps some gentle guidance could help put wayward ribosomes on the right track.

When you think of a chaperone, you probably think of a strict adult at the prom who keeps a tight rein on the kids’ behavior. Well, in nature, a chaperone sometimes has to do the opposite to help new genes form more quickly. Sometimes the chaperone has to give the gene a longer leash to explore lots of different possibilities.

Nature’s chaperones will look the other way when kids spike the punch.

See, in theory, it is pretty easy to make a new gene. A cell accidentally makes an extra copy of an existing gene and this gene is then free to mutate into something new. A few mutations later and you have a new gene.

Turns out this is probably trickier than it sounds. First off, having an extra copy of a gene can cause problems. And second, getting to a new function is no walk in the park either. It usually takes a few sequential mutations to get there and, with proteins being such persnickety things, many of the intermediates along the way end up being unstable.

One way a cell might deal with these issues is to bring in a chaperone that lets the gene tolerate more mutations. Chaperones are proteins that help stabilize other proteins, often under trying conditions like high temperature. They coddle the protein and keep it stable so that it can still do its job. In addition, chaperones can also cause a protein to relocate to different parts of the cell.

So the idea is that if a duplicated gene gains a mutation that lets its protein interact with a chaperone, the protein may get more stability from that interaction or may be rerouted to where it won’t do any harm. Because the chaperone buffers the possible harmful effects for the cell, the gene is free to explore more different intermediates on the way to its new function.

A new study out in GENETICS by Lachowiec and coworkers lends support to this “capacitor hypothesis.” The authors used both Arabidopsis and Saccharomyces cerevisiae to show that genes whose proteins interact with the chaperone Hsp90 evolved more quickly than closely related genes that did not. This strongly supports the idea that chaperones can encourage new functions in duplicated genes.

The authors first looked at a couple of closely related transcription factors from Arabidopsis, BES1 and BZR1. Using a specific inhibitor of HSP90 called geldanamycin (GdA), they were able to show that BES1 was a client of HSP90 but BZR1 was not. They then created a phylogenetic tree of ArabidopsisBZR/BEH gene family and, by determining the ratio of non-synonymous to synonymous changes, found that BES1 had a higher rate of mutation. One explanation is that the stabilizing/relocalizing influence of HSP90 allowed BES1 to tolerate more mutations.

This result was an excellent first step in showing that the capacitor hypothesis may be true in some cases, but it is limited by being based on a single pair of proteins. To broaden their findings, Lachowiec and coworkers took advantage of the vast knowledge about Hsp90 interactions in Saccharomyces cerevisiae to look at many more genes.

At first this didn’t work out that well. The authors looked at a data set of yeast proteins that interacted with Hsp90 (encoded in yeast by the HSP82 and HSC82 genes) and, after removing any co-chaperones from the set, found no difference in the rate of evolution between those proteins that interacted with Hsp90 and those that did not. But as the authors note, this isn’t surprising as so many other factors play a role in the rate of evolution too.

To refine their analysis, they mimicked their BES1/BZR1 study and focused on pairs of closely related proteins where one interacted with Hsp90 and the other did not. They found that proteins that interacted with Hsp90 had a “longer branch length” than did their close relatives that did not interact. In other words, Hsp90 appeared to help along the formation of a new gene.

The authors then went back to Arabidopsis and showed that BZR and BES1 were found in distinct but overlapping parts of the cell. This lends credence to the idea that chaperones cause proteins to localize to different parts of the cell.

So it looks like an important function of chaperones may be to shepherd new gene formation. They are more like a 1960’s version of a chaperone…they let duplicated genes make lots of mistakes on their way to discovering who they really are.